RESUMO
ß-barrel-shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell-wall of Gram-negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane-localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N-terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline-rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.
Assuntos
Anabaena/enzimologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/fisiologia , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Biossíntese de Proteínas , Transporte ProteicoRESUMO
Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a ß-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal ß-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.
Assuntos
Anabaena/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli , Congelamento , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms such as cyanobacteria, which are engineered to synthesize valuable products directly from CO2 and sunlight. For example, strains of the model organism Synechocystis sp. PCC 6803 have been generated to produce ethanol. Here, we performed a study to prove the hypothesis that carbon flux in the direction of pyruvate is one bottleneck to achieve high ethanol titers in cyanobacteria. Ethanol-producing strains of the cyanobacterium Synechocystis sp. PCC 6803 were generated that bear mutation in the gene pirC aiming to increase carbon flux towards pyruvate. The strains were cultivated at different nitrogen or carbon conditions and the ethanol production was analysed. Generally, a clear correlation between growth rate and ethanol production was found. The mutation of pirC, however, had only a positive impact on ethanol titers under nitrogen depletion. The increase in ethanol was accompanied by elevated pyruvate and lowered glycogen levels indicating that the absence of pirC indeed increased carbon partitioning towards lower glycolysis. Metabolome analysis revealed that this change in carbon flow had also a marked impact on the overall primary metabolism in Synechocystis sp. PCC 6803. Deletion of pirC improved ethanol production under specific conditions supporting the notion that a better understanding of regulatory mechanisms involved in cyanobacterial carbon partitioning is needed to engineer more productive cyanobacterial strains.
RESUMO
Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.
Assuntos
Anabaena/metabolismo , Porinas/genética , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Antibacterianos/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Metais/metabolismo , Mutação , Nitrogênio/metabolismo , Fenótipo , Porinas/metabolismo , Sais/metabolismo , Estresse Fisiológico/genéticaRESUMO
The introduction of alternative CO2-fixing pathways in photoautotrophic organism may improve the efficiency of biological carbon fixation such as minimizing the carbon loss due to photorespiration. Here, we analyzed the effects of creating a formate entry point into the primary metabolism of the cyanobacterium Synechocystis sp. PCC 6803. The formate-tetrahydrofolate ligase (FTL) from Methylobacterium extorquens AM1 was expressed in Synechocystis to enable formate assimilation and reducing the loss of fixed carbon in the photorespiratory pathway. Transgenic strains accumulated serine and 3-phosphoglycerate, and consumed more 2-phosphoglycolate and glycine, which seemed to reflect an efficient utilization of formate. However, labeling experiments showed that the serine accumulation was not due to the expected incorporation of formate. Subsequent DNA-microarray analysis revealed profound changes in transcript abundance due to ftl expression. Transcriptome changes were observed in relation to serine and glycine metabolism, C1-metabolism and particularly nitrogen assimilation. The data implied that ftl expression interfered with the signaling the carbon/nitrogen ratio in Synechocystis. Our results indicate that the expression of new enzymes could have a severe impact on the cellular regulatory network, which potentially hinders the establishment of newly designed pathways.